Organic cation rhodamines for screening organic cation transporters in early stages of drug development

• Published in: Journal of pharmacological and toxicological methods 2016-11, Vol.82, p.9-19
• Main Authors: Ugwu, Malachy C., Oli, Angus, Esimone, Charles O., Agu, Remigius U.
• Format: Article
• Language: English
• Subjects: Biological Transport; Calu-3 cells; Carnitine/organic cation transporter (OCTN1, OCTN2); Cell Line; Dose-Response Relationship, Drug; Drug Discovery - methods; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - pharmacokinetics; Humans; Models, Biological; Organic Cation Transport Proteins - agonists; Organic Cation Transport Proteins - antagonists & inhibitors; Organic Cation Transport Proteins - metabolism; Organic cation transporters (OCT1–3); Respiratory Mucosa - metabolism; Rhodamine 123 - chemistry; Rhodamine 123 - pharmacokinetics; Rhodamines; Rhodamines - chemistry; Rhodamines - pharmacokinetics; Sodium Azide - pharmacology
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2016.05.014

The aim of this study was to investigate the suitability of rhodamine-123, rhodamine-6G and rhodamine B as non-radioactive probes for characterizing organic cation transporters in respiratory cells. Fluorescent characteristics of the compounds were validated under standard in vitro drug transport conditions (buffers, pH, and light). Uptake/transport kinetics and intracellular accumulation of the compounds were investigated. Uptake/transport mechanisms were investigated by comparing the effect of pH, temperature, concentration, polarity, OCTs/OCTNs inhibitors/substrates, and metabolic inhibitors on the cationic dyes uptake in Calu-3 cells. Fluorescence stability and intensity of the compounds were altered by buffer composition, light, and pH. Uptake of the dyes was concentration-, temperature- and pH-dependent. OCTs/OCTNs inhibitors significantly reduced intracellular accumulation of the compounds. Whereas rhodamine-B uptake was sodium-dependent, pH had no effect on rhodamine-123 and rhodamine-6G uptake. Transport of the dyes across the cells was polarized: (AP→BL>BL→AP transport) and saturable: {Vmax=14.08±2.074, Km=1821±380.4 (rhodamine-B); Vmax=6.555±0.4106, Km=1353±130.4 (rhodamine-123) and Vmax=0.3056±0.01402, Km=702.9±60.97 (rhodamine-6G)}. The dyes were co-localized with MitoTracker®, the mitochondrial marker. Cationic rhodamines, especially rhodamine-B and rhodamine- 6G can be used as organic cation transporter substrates in respiratory cells. During such studies, buffer selection, pH and light exposure should be taken into consideration.